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Rheumatology Advance Access originally published online on July 27, 2004
Rheumatology 2004 43(11):1353-1356; doi:10.1093/rheumatology/keh349
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Rheumatology Vol. 43 No. 11 © British Society for Rheumatology 2004; all rights reserved

PAPER

Binding of high-avidity anti-ß2-glycoprotein I antibodies

S. Cucnik1, T. Kveder1, B. Rozman1 and B. Bozic1,2

1 Department of Rheumatology, University Medical Centre and 2 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia.

Correspondence to: B. Bozic, Department of Rheumatology, University Medical Centre, Vodnikova 62, 1000 Ljubljana, Slovenia. E-mail: borut.bozic{at}guest.arnes.si

Objectives. We studied the influence of different binding conditions on the interaction between high- or low-avidity IgG anti-ß2-glycoprotein I antibodies (anti-ß2-GPI) and ß2-GPI, which was either free in solution or bound to microtitre plates or nitrocellulose membranes.

Methods. Sera from 30 patients with antiphospholipid syndrome and/or systemic lupus erythematosus were selected on the basis of anti-ß2-GPI positivity. Avidity of IgG anti-ß2-GPI was determined by chaotropic ELISA, using increased NaCl concentration during the antibody binding. Immunodetection on nitrocellulose membrane followed reducing or non-reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (PAGE) of ß2-GPI. In converted, non-reducing PAGE, the preparation with high-affinity Fab fragments (obtained by papain digestion) was subjected to electrophoresis, while purified ß2-GPI was used as the sample in immunodetection.

Results. Anti-ß2-GPI antibodies of high, low or heterogeneous (low and high) avidity were found in 5/30, 9/30 and 16/30 sera, respectively. The density of ß2-GPI, which was 20–30 times higher on the nitrocellulose membrane than on the surface of ELISA plates, was not sufficient for the recognition of the antigen by anti-ß2-GPI: 2/5 high-avidity samples reacted only with non-reduced ß2-GPI, 3/9 low-avidity samples recognized only denatured and reduced ß2-GPI, and 1/16 samples with heterogeneous-avidity antibodies reacted with reduced and non-reduced ß2-GPI.

Conclusions. Our results suggest that neither high density of the antigen nor high avidity of the antibodies (or Fab fragments) alone was sufficient for the binding of anti-ß2-GPI to ß2-GPI. Some conformational modifications and, consequently, exposed neo-epitopes are required for the recognition of ß2-GPI by polyclonal anti-ß2-GPI antibodies.

KEY WORDS: Anti-ß2-glycoprotein I antibodies, Fab fragments, Avidity, Affinity, Antiphospholipid syndrome, Systemic lupus erythematosus


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